Ubiquitin, a small protein that acts as a post-translational mark on other proteins, has a surprising amount of conformational heterogeneity (Lange, O. F. et al. (2008) Science 320, 1471-1475). Protein ubiquitination mediates numerous cellular processes, such as cell cycle control, apoptosis, epigenetics and transcriptional regulation (Clague, M. J. et al (2010) Cell 143:682-685). The C terminus of ubiquitin is covalently attached to a lysine or the N terminus of a substrate protein through the coordinated action of three classes of enzymes: an E1 activator, an E2 conjugator and ubiquitin ligase (ubiquitin E3) (Pickart, C. M. (2001) Annu. Rev. Biochem. 70:503-533). Each of the seven lysines or the N terminus of ubiquitin can also serve as a substrate for further ubiquitination, allowing the polymerization of ubiquitin chains with linkages that convey a wealth of information (Pickart, C. M. et al (2004) Curr. Opin. Chem. Biol. 8:610-616). Motions within the β1-β2 loop of ubiquitin have been implicated in its recognition by partner proteins, though it is currently unknown how the conformational state of apo ubiquitin is ‘read’ by each partner. The multiple conformations of apo ubiquitin could represent a compromise that allows its single protein surface to bind a wide variety of unrelated partners with moderate affinity (Humphris, E. L. et al (2007) PLoS Comput. Biol. 3, e164 (2007); Friedland, G. D., et al (2009) PLoS Comput. Biol. 5, e1000393).
Deubiquitinases (DUBs) are a class of specialized proteases that regulate ubiquitin-mediated signaling by disassembling ubiquitin chains or removing monoubiquitination from substrates (Heideker, J. and Wertz, I. E. (2015) Biochem. J. 465:1-26; Lill, J. R. and Wertz, I. E. (2014) Trends in Pharm. Sci. 35(4):187-207; Komander, D., et al (2009) Nat. Rev. Mol. Cell Biol. 10:550-563). There are approximately 100 identified human DUBs, the majority of which await extensive investigation. One exception is USP7 (also known as HAUSP), a ubiquitin-specific protease (USP) which has an established role in tumorigenesis via its action on Mdm2, p53, FOXO4 and PTEN (Nicholson, B. et al (2011) Cell Biochem. Biophys. 60:61-68); Hussain, S., et al (2009) Cell Cycle 8:1688-1697). Although the determination of USP7's precise function in certain pathways has been complicated by conflicting reports (Li, M., et al (2004) Mol. Cell 13:879-886); Li, M. et al. (2002) Nature 416:648-653), this enzyme's participation in many cancer related processes has made it an attractive therapeutic target.
Despite its cellular importance, the catalytic core of USP7 is unexpectedly inactive, with a catalytic efficiency (kcat/Km) around 103 M-1 s-1 and an affinity (KD) for ubiquitin of several hundred micromolar (Faesen, A. C. et al. (2011) Mol. Cell 44:147-159); Fernández-Montalván, A. et al. (2007) FEBS J. 274, 4256-4270). The conformationally heterogeneous β1-β2 loop is directly contacted by all DUBs, including USP7. Hence, it is sought to determine whether stabilizing a USP7-binding conformation of ubiquitin's β1-β2 region could yield variants with a high affinity for the target DUB. Given USP7's role in cell division and technical difficulties surrounding USP7 knockdown, a conformationally optimized, ubiquitin-based inhibitor with high affinity and specificity for USP7 could prove a potent tool for understanding USP7's cellular functions. In a related approach, surface engineering of ubiquitin was used to generate potent binders of ubiquitin-signaling enzymes (Ernst et al, Science doi:10.1126/science. 1230161 (3 Jan. 2013); Zhang, Y., et al (2013) Nature Chemical Biology 9(1):51-58).
USP7 or HAUSP (herpesvirus-associated USP) is a ubiquitin specific protease or a deubiquitylating enzyme that cleaves ubiquitin from its substrates (Holowaty M N, et al (2003) J. Biol. Chem. 278 (48):47753-47756). Since ubiquitylation (polyubiquitination) is most commonly associated with the stability and degradation of cellular proteins, HAUSP activity generally stabilizes its substrate proteins. HAUSP is most popularly known as a direct antagonist of Mdm2, the E3 ubiquitin ligase for the tumor suppressor protein, p53 (Li M, et al (2002) Nature 416 (6881): 648-53). Normally, p53 levels are kept low in part due to Mdm2-mediated ubiquitylation and degradation of p53. In response to oncogenic insults, HAUSP can deubiquitinate p53 and protect p53 from Mdm2-mediated degradation, indicating that it may possess a tumor suppressor function for the immediate stabilization of p53 in response to stress. Another important role of HAUSP function involves the oncogenic stabilization of p53. Oncogenes such as Myc and E1A are thought to activate p53 through a p19 alternative reading frame (p19ARF, also called ARF)-dependent pathway, although some evidence suggests ARF is not essential in this process. A possibility is that HAUSP provides an alternative pathway for safeguarding the cell against oncogenic insults.
USP7 is a DUB that controls cell proliferation by altering the stability of Mdm2, p53, PTEN and FOXO4. Despite USP7's importance in tumor-related pathways, relatively little is known about its cellular and biochemical regulation, and there has been some confusion about its precise role in the Mdm2-p53 axis, in part because of a lack of inhibitory tools. The catalytic domain of USP7 is relatively inefficient in vitro, with a nearly undetectable affinity for its ubiquitin substrate and relatively poor catalytic efficiency. The C terminus and the fourth and fifth Ubl domains of USP7 greatly increase its activity via a combination of increased kcat and decreased Km, though the mechanism by which these effects are achieved is currently unknown. Engineering of ubiquitin conformation can greatly increase its interaction with deubiquitinases such as USP7 (Zhang, Y., et al (2013) Nature Chemical Biology 9(1):51-58).
Deregulation of the ubiquitin/proteasome system (UPS) has been implicated in the pathogenesis of many human diseases, including cancer (Vucic, D., et al (2011) Nature Reviews Molecular Cell Biology 12:439-452). Ubiquitin-specific proteases (USP) are cysteine proteases involved in the deubiquitination of protein substrates. Functional connections between USP7 and essential viral proteins and oncogenic pathways, such as the p53/Mdm2 and phosphatidylinositol 3-kinase/protein kinase B networks, strongly suggest that the targeting of USP7 with small-molecule inhibitors may be useful for the treatment of cancers and viral diseases.
Therapeutic agents that target apoptotic regulatory proteins that are part of the ubiquitin-proteasome system, might afford clinical benefits (Vucic, D., et al (2011) Nature Reviews Molecular Cell Biology 12:439-452). Deubiquitinase inhibitors and antagonists have been reported (Lill, J. R. and Wertz, I. E. (2014) Trends in Pharm. Sci. 35(4):187-207, see Table 2, page 195, Ndubaku, C. and Tsui, V. (2015) J. Med. Chem. DOI:10.1021/jm501061a). The rationale for targeting the ubiquitin-proteasome system for the treatment of cancer is validated by FDA approval, and the clinical efficacy, of bortezomib (VELCADE®; Millennium Pharmaceuticals) for the treatment of multiple myeloma and mantle cell lymphoma. Phenotypes associated with USP7 silencing strongly suggest that small molecule inhibitors of USP7 may have the potential for antiviral and anticancer therapies (Li, M. et al. (2002) Nature 416:648-653; Daviet, L. and Colland, F. (2008) Biochimie 90:270-283).